Esters of alkoxy-acylderivatives of carnitine and pharmaceutical compositions containing same

ABSTRACT

A novel class of esters of alkoxy-acylderivatives of carnitine, wherein the alkoxy-acyl radical has from 3 to 6 carbon atoms (typically: methoxyacetyl, methoxypropionyl, ethoxyacetyl, ethoxypropionyl and propoxyacetyl) is prepared by esterifying carnitine and then acylating the carnitine ester. Such esters are therapeutically useful in the treatment of cardiac disorders, hyperlipidaemias and hyperlipoproteinaemias.

This is a continuation of co-pending application Ser. No. 263,655 filedon May 14, 1981 now abandoned.

The present invention relates to a novel class of esters ofalkoxy-acylderivatives of carnitine, the process for their preparation,the pharmaceutical compositions containing such esters and their use intherapy.

More particularly, the present invention relates to esters ofalkoxy-acylderivatives of carnitine having general formula (I): ##STR1##wherein: X⁻ is an halogen anion selected between chlorine and bromine,preferably chlorine;

R is a alkoxy-acyl radical having from 3 to 6 carbon atoms, such radicalbeing preferably selected from the group consisting of methoxyacetyl, 2-and 3- methoxypropionyl, ethoxyacetyl, 2- and 3- ethoxypropionyl andpropoxyacetyl;

R' is an alkyl radical having from 1 to 6 carbon atoms, such radicalbeing preferably selected from the group consisting of methyl, ethyl,propyl, isopropyl, butyl and isobutyl.

It should be understood that the foregoing general formula (I)encompasses the esters of the present invention both in their opticallyactive forms and in their racemic form.

It has been found that the compounds of the present invention possessvaluable pharmacological properties and can, therefore, be utilized inthe therapeutical field.

More particularly: The esters of formula (I) showed to be endowed with aprolonged and effective inotropic action and devoid of any effectsdepressing the myocardial excitability. Although no theoreticalinterpretation is intended here, it is deemed that the foregoing is dueto the capability of the ester bond of protecting acyl-carnitine againstrapid metabolic degradation and sharp fall of blood levels thereof.

Moreover, the esters of formula (I) have shown to be endowed with anantifibrillation effect.

The direct antiarrhythmic effect of the quinidine type is complementedby the adrenaline-antagonizing activity.

The esters of formula (I) have been shown to restore the α- andβ-lipoprotein ratio to normal.

The compounds of this invention can, therefore, be therapeuticallyutilized

(a) in cases of myocardial hypocontractility, such as in cardiogenicshock provoked by primary absence of contractile force;

(b) for the treatment of functional arrhythmias and arrhythmiassecondary to myocardial-sclerotic processes; and

(c) for the treatment of hyperlipidaemias and hyperlipoproteinaemias.

The process for preparing the esters of alkoxy-acylderivatives ofcarnitine in accordance with the present invention comprises the stepsof:

(a) esterifying carnitine by suspending carnitine in an alcohol havingformula R'OH wherein R' has the previously specified meaning, bubblinginto the resulting suspension gaseous hydrochloric acid till completedissolution of carnitine, refluxing the resulting solution and isolatingthe carnitine ester by concentration and neutralization of saidsolution; and

(b) acylating the carnitine ester of step (a) by reacting it with anexcess of acyl halogenide of formula RX wherein R has the previouslyspecified meaning and X is an halogen atom selected between chlorine andbromine, keeping the resulting reaction mixture for about 42-80 hours atabout 35°-60° C. and isolating the ester of alkoxy-acyl carnitine offormula (I) by means of conventional procedures.

In step (b) the carnitine ester of step (a) can be dissolved in aninert, anhydrous organic solvent and the reaction mixture be kept in astream of inert, anhydrous gas. Alternatively, the carnitine ester ofstep (a) can be reacted with an excess of acylating agent in the absenceof solvent and the reaction mixture be kept in a moisture-freeenvironment.

In case the carnitine ester is dissolved in a solvent, the solvent ispreferably selected among acetone, methylene chloride, chloroform andacetonitrile.

As mentioned, the carnitine ester is reacted with an excess of anacylating agent. Preferably, the molar ratio between the carnitine esterand the acylating agent is comprised between 1:2 and 1:4.

The following non limiting examples aim at illustrating the preparationof some esters of alkoxy-acylderivatives of carnitine in accordance withthe present invention.

EXAMPLE 1 Preparation of methoxyacetyl carnitine isobutyl ester

(1) Preparation of carnitine isobutyl ester: Carnitine hydrochloride (10g; 0.05 moles) was suspended in 100 ml of isobutanol. The resultingmixture was cooled with an ice bath and gaseous hydrochloric acid wasbubbled therein till complete saturation. The resulting mixture was keptunder reflux conditions for 2 hours. The mixture was then concentratedand subsequently taken up with isobutanol and neutralized with Amberlist21. The resulting solution was then filtered and brought to dryness. 12g of carnitine isobutyl ester were obtained.

(2) Preparation of methoxy-acetyl chloride: SOCl₂ (1.1 cc; 0.0125 moles)was added to methoxy-acetic acid (1.08 g; 0.012 moles). The resultingreaction mixture was kept at room temperature for 12 hours and was thenwashed three times with a mixture of chloroformanhydrous ethyl ether andsubsequently concentrated under vacuum (P=80 mm Hg) at 30° C. 1.2 g ofmethoxy-acetyl chloride were obtained.

(3) Reaction between carnitine isobutyl ester and methoxy-acetylchloride: Carnitine isobutyl ester (1.1 g; 0.0043 moles) prepared asindicated in step (1) was dissolved in anhydrous acetone and to theresulting solution the methoxy-acetyl chloride (970 mg; 0.009 moles)which had been prepared as indicated in step (2) was added. The reactionmixture was brought to dryness and the residue kept in an atmosphere ofinert gas (nitrogen and argon) at 40° C. for 48 hours. Subsequently, theresidue was crystallized from isopropanol-ethyl ether.

The title product was obtained with a yield of 55%. TLC Eluant: CHCl₃,MeOH, CH₃ COONa 0.01M 40,40,10.

    ______________________________________                                        NMR D.sub.2 O δ                                                                      ##STR2##                                                                     4.2 (2H, s, COOCH.sub.2);                                                      ##STR3##                                                                     3.4 (3H, s, OCH.sub.3);                                                        ##STR4##                                                                     2.9 (2H, d, CH.sub.2 COO);                                                     ##STR5##                                                                      ##STR6##                                                         ______________________________________                                    

EXAMPLE 2 Preparation of ethoxyacetyl carnitine isobutyl ester

(1) Preparation of carnitine isobutyl ester: Carnitine hydrochloride (10g; 0.05 moles) was suspended in 100 ml of isobutanol. The resultingsuspension was cooled with an ice bath and gaseous hydrochloric acid wasbubbled therein till complete saturation. The resulting mixture was keptunder reflux conditions for 2 hours. The mixture was concentrated inorder to remove the alcohol; the concentrate was dissolved in distilledwater and neutralized with IR 45 resin. The resulting product waslyophilized, thus obtaining 12 g of carnitine isobutyl ester.

(2) Preparation of ethoxy acetyl chloride: To ethoxy acetic acid (1.3cc; 0.012 moles) thionyl chloride (1.1 cc; 0.0125 moles) was added. Theresulting mixture was kept at room temperature for 12 hours. Thereaction mixture was washed three times with a mixture ofchloroform-anhydrous ethyl ether and subsequently concentrated undervacuum (80 mm Hg) at 30° C. 1.15 g of ethoxy acetyl chloride wereobtained.

(3) Reaction between carnitine isobutyl ester and ethoxy acetylchloride: Carnitine isobutyl ester (1.1 g; 0.043 moles) was dissolved inanhydrous acetone and to the resulting solution ethoxy acetyl chloride(1.15 g; 0.009 moles) was added. The reaction mixture was brought todryness and the residue kept in an atmosphere of inert gas (argon) atroom temperature for 2 days.

Subsequently, the residue was crystallized from isopropanol-ethyl ether.The title product was obtained with a yield of 65%.

    ______________________________________                                        TLC Eluant:                                                                            CHCl.sub.3 40                                                                 CH.sub.3 OH                                                                              40                                                                 CH.sub.3 COONa                                                                           0.01M 10                                                  NMR                                                                                     ##STR7##                                                                     4.2 (2H, s, COCH.sub.2 O);                                                    4.0 (4H, m, COOCH.sub.2, O ---CH.sub.2 CH.sub.3);                              ##STR8##                                                                     2.7 (2H, d, CH.sub.2 COO);                                                     ##STR9##                                                                      ##STR10##                                                           ______________________________________                                    

EXAMPLE 3 Preparation of 3-ethoxypropionyl carnitine isopropyl ester

(1) Preparation of carnitine isopropyl ester: Carnitine hydrochloride(10 g; 0.05 moles) was suspended in 100 cc of isopropanol. The resultingsuspension was cooled with ice bath and gaseous hydrocloric acid wasbubbled therein till complete saturation. Subsequently the mixture washeated under reflux conditions for 2 hours. The mixture was thenconcentrated, subsequently taken up with isopropanol and neutralizedwith Amberlist 21. The resulting solution was filtered and brought todryness. 11 g of carnitine isopropyl ester were obtained.

(2) Preparation of 3-ethoxy propionyl chloride: To 3-ethoxy propionicacid (1.18 g; 0.01 moles) oxalyl chloride (3.78 g; 0.03 moles) wasadded. The resulting mixture was kept under stirring at room temperaturefor 4 hours. Subsequently, the resulting mixture was washed three timewith anhydrous ethyl ether and concentrated under vacuum (P=90 mmHg) at40° C. 1.2 g of 3-ethoxy propionyl chloride were obtained.

(3) Reaction between carnitine isopropyl ester and 3-ethoxy propionylchloride: Carnitine isopropyl ester (1.1 g; 0.0045 moles) obtained inthe previous step (1) was dissolved in anhydrous methylene chloride andto the resulting solution 3-ethoxy propionyl chloride (1.2 g; 0.009moles) obtained in the previous step (2) was added. An inert gas(nitrogen) was bubbled into the reaction mixture till completeevaporation of the solvent. Subsequently, the mixture was kept at 40° C.for 24 hours. The residue was crystallized fromisopropanol-aceton-ether. The product was obtained with a yield of 60%.TLC Eluant: chloroform, methanol, sodium acetate 0.01M 40/40/10

    ______________________________________                                        NMR D.sub.2 O δ                                                                    ##STR11##                                                                    5.0 (1H, m, COOCH);                                                            ##STR12##                                                                    3.3 (9H, s, .sup.+N(CH.sub.3).sub.3);                                         2.9 (2H, d, CH ---CH.sub.2 COO);                                              2.5 (2H, t, OCO ---CH.sub.2 CH.sub.2);                                         ##STR13##                                                          ______________________________________                                    

EXAMPLE 4 Preparation of 2-methoxy propionyl carnitine isopropyl ester

Preparation of the 2-methoxy propionic acid: 2-bromo propionic acid(30.4 g; 0.2 moles) was added to a solution of 10 g of metal sodium in150 ml of anhydrous methanol. The resulting mixture was kept understirring at 60° C. for 3 days. Subsequently, the solution wasconcentrated under vacuum, the residue was taken up with water,acidified with conc. HCl and the resulting aqueous solution wasextracted with ethyl ether. The organic phase, washed with water anddried on Na₂ SO₄, was concentrated under vacuum. Pure 2-methoxypropionic acid was thus obtained, as confirmed by NMR analysis.

(2) Preparation of 2-methoxy propionyl chloride: 2-methoxy propionicacid (3.128 g; 0.03 moles) and thionyl chloride (2.3 g; 0.04 moles) werekept under stirring at 50° C. for 4 hours in anhydrous environment. Theexcess of thionyl chloride was removed under vacuum and the raw materialthus obtained was utilized in the subsequent reaction.

(3) Preparation of 2-methoxy propionyl carnitine isopropyl ester:Carnitine isopropyl ester (2.4 g; 0.01 moles) prepared as previouslydescribed and 2-methoxy propionyl chloride were reacted at 50° C. for 3days. The reaction mixture was treated with ethyl ether, the rawmaterial which precipitated was taken up with acetonitrile anddecolorized with activated carbon.

To the acetonitrile solution ethyl ether was added, thus obtaining aprecipitate which, upon examination with HPLC (column C₁₈ : eluant NH₄H₂ PO₄ --CH₃ CN 85-15, flow rate 1 ml/min. RI detector) turned out toconsist of the product mixture. The mixture was purified withpreparative HPLC: CHROMATOSPAS PREP 100, column pressure 6 Barr; eluantpressure 5 Barr; flow rate 15 ml/min; lichroprep RP8 resin, diameter3.60μ. The product thus obtained was shown to consist of 2-methoxypropionyl carnitine isopropyl ester. Yield 50%.

    ______________________________________                                        NMR D.sub.2 O δ                                                                   ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                    2.9 (2H, d, CH.sub.2 COO);                                                    1.4 (3H, d, CH ----CH.sub.3);                                                  ##STR17##                                                           ______________________________________                                    

PHARMACOLOGICAL ACTIVITIES

The pharmacological properties of the compounds of the present inventionwere investigated with the following techniques:

(a) Acute toxicity (LD50)

Acute toxicity was investigated by using the method disclosed by Weil C.S. in "Tables for convenient calculation of median-effective dose (LD50or ED50) and instructions in their use", Biometrics, 249-253, 1952.

The tolerance of the compounds under examination was investigated inmice after administration by the intraperitoneal or oral route. Theobtained results show that the compounds exhibit excellent tolerance.(see the Table).

(b) Inotropic effect

Rabbit hearts isolated by the Langendorff method were perfused withoxygenized Ringer solution at 38.2° C. The isometric contractions,electrogardiogram and coronary flow were recorded using a"Battaglia-Rangoni" polygraph. By removing the oxygen from the perfusionfluid, metabolic damage was induced in the cardiac muscle, up to an 80%reduction in the cardiac contractile force.

Under these conditions of prolonged anoxia the aerobic glycolysis of themyocardium is slowed down, accompanied by the storage of acidcatabolites due to both the accumulation of pyruvic acid and itsconversion to lactic acid which cannot be utilized because of thedepression of pyridine enzymes, such as LDH (lactodehydrogenase). Thishas repercussions on the anaerobic glycolysis affecting an everincreasing number of enzymes, accompanied by a progressive andincreasingly critical exhaustion of the myocardium. Thus a whole seriesof cardiac muscle fatigue levels occurs which can be observed by thebehavior of the examined parameters, namely the contractile force,coronary flow, heart rate and cardiac rhythm. As soon as the contractileforce was reduced by 80%, the perfusion fluid was once again oxygenizedeither without adding other compounds (controls) or with the addition ofthe compounds under examination.

The contractile force of the heart was examined, which shows a positiveinotropic effect after 10 minutes from the interruption of the anoxicperiod (myocardial restoration). The results, evaluated by means ofStudent's "t" test, show that the compounds under examination induce apositive inotropic effect statistically significant against thecontrols.

In the Table there are shown the percentage value of increase againstthe controls.

(c) Antiarrhythmic effect

In order to evaluate the antiarrhythmic activity of the carnitinederivatives of this invention studied with in vivo tests in addition toand in comparison with the currently employed in vitro tests, the methoddisclosed by Nwangwu et al. (Arch. Int. Pharmacodyn., 1977, 229, 219)was used.

According to this method an aconitine solution is injected into thecaudal vein of mice and the onset time of arrhythmia and tachycardiaafter 2 to 60 minutes from administration of the compounds underexamination is recorded.

The antiarrhythmic activity calculated from the increase in the latencytime of the onset of the arrhythmias of the treated animals incomparison with the controls, is illustrated in the Table.

(d) Adrenaline-antagonizing effect

Groups of ten male Albino Swiss mice, weighing 12-22 g, wereintraperitoneally administered either with the esters of the presentinvention or with saline (control) and, after 30 minutes, withadrenaline (treated) at a dose capable of bringing about death to 100%of the control animals due to ventricular fibrillation and cardiaclesions ensuing from increase in frequency, pressure and oxygen uptakefrom the myocardium.

Mortality was checked for 36 hours and the effect of the compoundsexpressed as percentage of surviving animals, is shown in the Table.

                                      TABLE                                       __________________________________________________________________________    Pharmacological activity of some esters of alkoxy-acyl derivatives of         carnitine.                                                                    LD50 by the intraperitoneal route in mice, antifibrillatory activity in       mice, adrenaline-antagonizing                                                 activity in mice, inotropic activity on rabbit isolated heart.                 ##STR18##             i.p.LD50 mg kg.sup.-1                                                                 % reduction(dose mg kg.sup.-1                                                i.v.)Antifibrillatory activity                                                             % mortality reduction(dose mg                                                kg.sup.-1 i.p.)Antiadrenaline                                                 activity     % of controls(dose                                                           10.sup.-5                                                                     gl.sup.-1)notropic                                                            effect                  __________________________________________________________________________    R = methoxyacetyl;                                                                          R' = methyl                                                                           1200     75 (300)   70 (450)    +72                                   R' = ethyl                                                                            550     70 (50)     70 (70)     +58                                   R' = propyl                                                                           650     100 (50)    75 (50)     +78                                   R' = isopropyl                                                                        800     80 (50)     75 (50)     +55                                   R' = butyl                                                                            550     75 (40)     70 (100)    +55                                   R' = isobutyl                                                                         690     85 (75)     60 (300)    +79                     R = ethoxyacetyl;                                                                           R' = methyl                                                                           1100     90 (150)   75 (300)    +55                                   R' = ethyl                                                                            600     65 (35)     50 (50)     +68                                   R' = propyl                                                                           620     75 (50)     75 (100)    +49                                   R' = isopropyl                                                                        700     70 (50)     60 (100)    +72                                   R' = butyl                                                                            600     75 (40)     75 (100)    +58                                   R' = isobutyl                                                                         750     70 (10)     50 (70)     +70                     R = 2-ethoxypropionyl;                                                                      R' = isopropyl                                                                        1200    70 (50)     50 (300)    +50                     R = 2-methoxypropionyl;                                                                     R' = isopropyl                                                                        850     68 (50)     58 (100)    +70                     __________________________________________________________________________

The compounds of the present invention are administered either orally orparenterally, in any of the usual pharmaceutical forms which areprepared by conventional procedures, well known to the experts in thepharmaceutical field. These forms comprise solid and liquid oral unitdosage forms, such as tablets, capsules, solutions, syrups and the likeand injectable forms such as sterile solutions for ampoules and vials.

For preparing such pharmaceutical forms the usual solvents, diluents andexcipients are used. Optionally, preservative, sweetening and flavoringagents can also be present. Non limiting examples of such substances aresodium carboxymethyl cellulose, polysorbate, mannitol, sorbitol, starch,avicel, talc and other substances which will be apparent to the expertsof pharmaceutical techniques.

The dose to be administered will be determined by the attendingphysician taking the age, weight and general conditions of the patientinto account, utilizing sound professional judgment. Although effectiveresults can be observed even at doses as low as from 5 to 8 mg/kg ofbody weight daily, a daily dose of from about 10 to about 50 mg/kg ofbody weight is preferred. Should it be deemed necessary, larger dosescan be administered, in view of the low toxicity of the compounds ofthis invention.

Non-limiting example of dosages are as follows:

fials: 5-500 mg

capsules: 15-50 mg

tablets: 15-500 mg

oral solutions: 15-50 mg

What is claimed is:
 1. An ester of alkoxy-carboxyl derivatives ofcarnitine having general formula (I): ##STR19## wherein: X⁻ is a halogenanion selected from chlorine and bromine;R is an alkoxy-substitutedalkanoyl group radical having from 3 to 6 carbon atoms; and R' is analkyl radical having from 1 to 6 carbon atoms.
 2. An ester according toclaim 1, wherein said radical R is selected from the group consisting ofmethoxyacetyl, 2- and 3-methoxypropionyl, ethoxyacetyl, 2- and3-ethoxypropionyl and propoxyacetyl, and said alkyl radical R' isselected from the group consisting of methyl, ethyl, propyl, isopropyl,butyl and isobutyl.
 3. A pharmaceutical composition for the treatment ofcardiac disorders, hyperlipoproteinaemias, or hyperlipidaemias,comprising a therapeutically effective amount of an ester of carnitineof formula (I) ##STR20## wherein: X⁻ is a halogen anion selected fromchlorine and bromine;R is an alkoxy-substituted alkanoyl radical havingfrom 3-6 carbon atoms; and R' is an alkyl radical having from 1 to 6carbon atoms, and a pharmacologically acceptable excipient.